Heart disease is the major cause of death in the United States. A loss of myocardial function, or failure to replace damaged myocardium, underlies the tremendous burden on our society caused by cardiovascular disease, killing an average of 1 person every 39 seconds. Likewise, cardiovascular developmental anomalies are the most common congenital defects in newborns, presenting in nearly 1% of live births. Many of the transcription factors controlling cardiogenesis are now known and comprise a molecular network essential for normal heart growth, morphogenesis, and function. However, the initial step of specifying cardiac progenitors from uncommitted early embryonic mesoderm is poorly understood. Progress in understanding this process could be exploited to develop regenerative strategies for treating cardiovascular disease, including heart failure. We have discovered a genetic pathway that is both necessary and sufficient during embryonic development for the generation of cardiomyocytes. A central component of this pathway is the Gata5 transcription factor, which has been relatively overlooked with respect to its function in cardiogenesis. We found that expression of Gata5, during a specific developmental window, is sufficient to efficiently generate cardiomyocytes from a mouse pluripotent stem cell progenitor population. We also showed that gata5 is required during embryogenesis, along with its sister gene gata6, for the normal specification of zebrafish cardiomyocytes. Thus, we established high-throughput model systems to generate or eliminate the production of cardiomyocytes. We developed tools to discover the genetic pathways controlled by gata5 at both the transcriptional and translational levels. We developed new conditional strategies to control the pathway with spatial and temporal specificity. We have assembled a team with expertise in genome-wide molecular network modeling, and we can evaluate the function of candidate network components with high throughput. We propose to fully interrogate at the whole genome level the essential downstream molecular pathway(s) that promote or restrict the generation of cardiomyocytes, and to discover novel regulators of cardiomyocyte fate. A complete understanding of these programs will reveal new cellular or pharmacological strategies for restoring damaged cardiac tissue caused by cardiovascular disease. Toward this goal, Aims are proposed to: 1) Discover the genetic network sufficient to mediate cardiomyocyte specification in a mammalian embryonic stem cell model. 2) Identify the regulatory network necessary to mediate cardiomyocyte specification in a vertebrate embryo, using a complementary zebrafish animal model, and 3) Define temporal- specific components of the cardiomyocyte specification program using conditional loss-of-function strategies in the zebrafish model. Most importantly, we will identify and test the function of previously unknown components of the cardiomyocyte specification network.